Protocol for quantifying <scp>miRNA</scp> trafficking across the endosomal membrane

The import of miRNA across the endosomal membrane is an essential prerequisite for the loading of miRNAs into the endosomal lumen. These miRNAs are then incorporated into mature multivesicular bodies (MVBs), which then fuse with the plasma membrane, leading to the release of exosomes or extracellular vesicles carrying miRNAs into the intercellular space. Here, we present a reproducible in vitro assay for the quantification of miRNA import across the endosomal membrane. We detail the isolation of intact endosomes from mammalian cells, followed by the loading of synthetic, chemically modified microRNAs (miRNAs) into the vesicles in an RNase-resistant form. Integrating differential centrifugation and iodixanol density-gradient fractionation, we isolate endosomes free of endoplasmic reticulum contamination. Then, we use optimized in vitro cargo-loading techniques to facilitate subsequent assessment of miRNA uptake, stability, and delivery efficiency in the presence of altered external factors, such as ATP or substrate concentration. Ultimately, this protocol presents a standardized method for quantifying miRNA trafficking across the endosomal membrane.